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Screening and large-scale expression of membrane proteins in mammalian cells for structural studies

Nature protocols, 2014-11, Vol.9 (11), p.2574-2585 [Peer Reviewed Journal]

COPYRIGHT 2014 Nature Publishing Group ;COPYRIGHT 2014 Nature Publishing Group ;Copyright Nature Publishing Group Nov 2014 ;Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. 2014. ;ISSN: 1754-2189 ;EISSN: 1750-2799 ;DOI: 10.1038/nprot.2014.173 ;PMID: 25299155

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Title:
Screening and large-scale expression of membrane proteins in mammalian cells for structural studies
Author: Goehring, April ; Lee, Chia-Hsueh ; Wang, Kevin H ; Michel, Jennifer Carlisle ; Claxton, Derek P ; Baconguis, Isabelle ; Althoff, Thorsten ; Fischer, Suzanne ; Garcia, K Christopher ; Gouaux, Eric
Subjects: Acid Sensing Ion Channels - genetics ; Acid Sensing Ion Channels - metabolism ; Animals ; Baculovirus ; Caenorhabditis elegans Proteins - genetics ; Caenorhabditis elegans Proteins - metabolism ; Cell culture ; Chickens ; Chloride Channels - genetics ; Chloride Channels - metabolism ; Chloride ions ; Chromatography, Gel ; Crystallography ; Fluorescence ; Genetic Vectors ; Green Fluorescent Proteins - genetics ; Green Fluorescent Proteins - metabolism ; HEK293 Cells ; Histidine - genetics ; Humans ; Ion channels ; Mammalian cells ; Mammals ; Membrane proteins ; Membrane Proteins - genetics ; Membrane Proteins - metabolism ; Membranes ; Methods ; N-Acetylglucosaminyltransferases - metabolism ; N-Acetylglucosinyldiphosphodolichol N-acetylglucosaminyltransferase ; Plasmids - genetics ; Properties ; Protein biosynthesis ; Protein Engineering - methods ; Protein research ; Proteins ; Recombinant Proteins - genetics ; Recombinant Proteins - metabolism ; Screening ; Size exclusion chromatography ; Structural analysis ; Suspension culture ; Transfection ; Transfection - methods ; X-ray crystallography
Is Part Of: Nature protocols, 2014-11, Vol.9 (11), p.2574-2585
Description: Structural, biochemical and biophysical studies of eukaryotic membrane proteins are often hampered by difficulties in overexpression of the candidate molecule. Baculovirus transduction of mammalian cells (BacMam), although a powerful method to heterologously express membrane proteins, can be cumbersome for screening and expression of multiple constructs. We therefore developed plasmid Eric Gouaux (pEG) BacMam, a vector optimized for use in screening assays, as well as for efficient production of baculovirus and robust expression of the target protein. In this protocol, we show how to use small-scale transient transfection and fluorescence-detection size-exclusion chromatography (FSEC) experiments using a GFP-His8-tagged candidate protein to screen for monodispersity and expression level. Once promising candidates are identified, we describe how to generate baculovirus, transduce HEK293S GnTI(-) (N-acetylglucosaminyltransferase I-negative) cells in suspension culture and overexpress the candidate protein. We have used these methods to prepare pure samples of chicken acid-sensing ion channel 1a (cASIC1) and Caenorhabditis elegans glutamate-gated chloride channel (GluCl) for X-ray crystallography, demonstrating how to rapidly and efficiently screen hundreds of constructs and accomplish large-scale expression in 4-6 weeks.
Publisher: England: Nature Publishing Group
Language: English
Identifier: ISSN: 1754-2189
EISSN: 1750-2799
DOI: 10.1038/nprot.2014.173
PMID: 25299155
Source: MEDLINE
ProQuest Central

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Screening and large-scale expression of membrane proteins in mammalian cells for structural studies

COPYRIGHT 2014 Nature Publishing Group ;COPYRIGHT 2014 Nature Publishing Group ;Copyright Nature Publishing Group Nov 2014 ;Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. 2014. ;ISSN: 1754-2189 ;EISSN: 1750-2799 ;DOI: 10.1038/nprot.2014.173 ;PMID: 25299155

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